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Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges from 4.7 to 7.4, and temperature from 10°C to 37°C for B. weihenstephanensis and from 18°C to 59°C for B. licheniformis. Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits, was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat-treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C during 12 min or at 95°C during 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite they both led to a tenfold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth by 1.2 fold and lowered the proportion of spores able to germinate and outgrow by 1.4 fold. Interestingly, we showed by a proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 compared to those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH. Importance Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sub-lethal heat-treatment. Using flow cytometry we were able to follow at a single cell level the changes in the physiological states of heat-stressed spores of Bacillus sp. and to discriminate between dormant spores, germinated spores and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth, and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sub-lethal heat-treatment, a common situation in food processing and a concern for food preservation and safety.

Geobacillus stearothermophilus spores are recognized as one of the most wet-heat resistant among aerobic spore-forming bacteria and are responsible for 35% of canned food spoilage after incubation at 55 °C. The purpose of this study was to investigate and model the fate of heat-treated survivor spores of G. stearothermophilus ATCC 12980 in growth-preventing environment. G. stearothermophilus spores were heat-treated at four different conditions to reach one or two decimal reductions. Heat-treated spores were stored in nutrient broth at different temperatures and pH under growth-preventing conditions. Spore survival during storage was evaluated by count plating over a period of months. Results reveal that G. stearothermophilus spores surviving heat treatment lose their viability during storage under growth-preventing conditions. Two different subpopulations were observed during non-thermal inactivation. They differed according to the level of their resistance to storage stress, and the proportion of each subpopulation can be modulated by heat treatment conditions. Finally, tolerance to storage stress under growth-preventing conditions increases at refrigerated temperature and neutral pH regardless of heat treatment conditions. Such results suggest that spore inactivation due to heat treatment could be completed by storage under growth-preventing conditions.

Essential oil was obtained in a yield 1.1%, w/w, by steam distillation of Elionurus tristis leaves from Madagascar. The chemical composition was analyzed qualitatively and quantitatively by GC-MS and GC-FID, respectively. To the best of our knowledge, this is the first chemical analysis of this essential oil. Seventy-three compounds were identified, corresponding to 94.9% of the total essential oil. The principal compounds were sesquiterpenes and the more represented were β-gurjunene (18.4%), neoclovene (15.8%) and nootkatone (10.4%). Through a comparative study, we observed a large variability between the components of E. tristis essential oil and those from others species of the same genus. Evaluation of the antioxidant (ABTS and DPPH assays) and anti-tuberculosis activities of the essential oil showed weak antioxidant potency but an interesting anti-tuberculosis activity with a MIC of 32 mg/L. This activity prompted us to evaluate individually the major components for the treatment of tuberculosis.

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis ) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.

The objective of this study was to evaluate acceptability and instrumental measurements (titratable acidity, total soluble sugar, total phenolic, flavonoids, anthocyanins, and condensed tannin contents as well as antioxidant capacity) of seven Tunisian pomegranate cultivars to relate fruit acceptability and beneficial health. Seventy one participants were asked to complete a questionnaire and to indicate their level of acceptance, on a nine point-hedonic scale, for fruit size, sweetness, bitterness, juice color and juice overall acceptability. Taste and aril color were the main criteria to select Pomegranate. Significant differences occurred for all the acceptability tests. Titratable acidity determines the taste and important inter-specific variability in the phenolic contents as well as for the antioxidant capacities was noticed. Anthocyanin and condensed tannin were contributing to the organoleptic properties while bitterness acceptability was negatively correlated to the antioxidant capacity. Also, a relationship was found between the fruit size acceptability and the antioxidant capacity.

Although heat treatment is probably the oldest and the most common method used to inactivate spores in food processes, the specific mechanism of heat killing of spores is still not fully understood. The purpose of this study is to investigate the evolution of the permeabilization and the viability of heat-treated spores during storage under growth-preventing conditions. Geobacillus stearothermophilus spores were heat-treated under various conditions of temperature and pH, and then stored under conditions of temperature and pH that prevent growth. Spore survival was evaluated by count plating immediately after heat treatment, and then during storage over a period of months. Flow cytometry analyses were performed to investigate the Syto 9 permeability of heat-treated spores. Sub-lethally heat-treated spores of G. stearothermophilus were physically committed to permeabilization after heat treatment. However, prolonged heat treatment may abolish the spore permeabilization and block heat-treated spores in the refractive state. However, viability loss and permeabilization during heat treatment seem to be two different mechanisms that occur independently, and the loss of permeabilization properties takes place at a much slower rate than spore killing. Under growth-preventing conditions, viable heat-treated spores presumably lose their viability due to the permeabilization phenomena, which makes them more susceptible to the action of adverse conditions precluding growth.

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Thermophilic spore-forming bacteria are potential contaminants in several industrial sectors involving high temperatures (40-65 °C) in the manufacturing process. Among those thermophilic spore-forming bacteria, Thermoanaerobacterium thermosaccharolyticum, called "the swelling canned food spoiler", has generated interest over the last decade in the food sector. The aim of this study was to investigate and to model pH effect on growth, heat resistance and recovery abilities after a heat-treatment of T. thermosaccharolyticum DSM 571. Growth and sporulation were conducted on reinforced clostridium media and liver broth respectively. The highest spore heat resistances and the greatest recovery ability after a heat-treatment were obtained at pH condition allowing maximal growth rate. Growth and sporulation boundaries were estimated, then models using growth limits as main parameters were extended to describe and quantify the effect of pH on recovery of injured spores after a heat-treatment. So, cardinal values were used as a single set of parameters to describe growth, sporulation and recovery abilities. Besides, this work suggests that T. thermosaccharolyticum preserve its ability for germination and outgrowth after a heat-treatment at a low pH where other high resistant spore-forming bacteria like Geobacillus stearothermophilus are unable to grow.

Geobacillus stearothermophilus is recognized as one of the most prevalent micro-organism responsible for flat sour in the canned food industry. To control these highly resistant spore-forming bacteria, the heat treatment intensity could be associated with detrimental conditions for germination and outgrowth. The purpose of this work was to study successively the impact of temperature and pH on the growth rate of G. stearothermophilus ATCC 12980, its sporulation ability, its heat resistance in response to various sporulation conditions, and its recovery ability after a heat treatment. The phenotypic investigation was carried out at different temperatures and pHs on nutrient agar and the heat resistance was estimated at 115 °C. The greatest spore production and the highest heat resistances were obtained at conditions of temperature and pH allowing maximal growth rate. The current observations also revealed that growth, sporulation and recovery boundaries are close. Models using growth boundaries as main parameters were extended to describe and quantify the effect of temperature and pH throughout the life cycle of G. stearothermophilus as vegetative cells or as spore after a heat treatment and during recovery.

In Saccharomyces cerevisiae, synthesis of T6P (trehalose 6-phosphate) is essential for growth on most fermentable carbon sources. In the present study, the metabolic response to glucose was analysed in mutants with different capacities to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1, which had no measurable T6P, exhibited impaired ethanol production, showed diminished plasma membrane H+-ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted into inosine. Deletion of the AMP deaminase encoding gene, AMD1, in the tps1 strain prevented inosine formation, but did not rescue energy balance or growth on glucose. Neither the 90%-reduced T6P content observed in a tps1 mutant expressing the Tps1 protein from Yarrowia lipolytica, nor the hyperaccumulation of T6P in the tps2 mutant had significant effects on fermentation rates, growth on fermentable carbon sources or plasma membrane H+-ATPase activation. However, intracellular metabolite dynamics and pH homoeostasis were strongly affected by changes in T6P concentrations. Hyperaccumulation of T6P in the tps2 mutant caused an increase in cytosolic pH and strongly reduced growth rates on non-fermentable carbon sources, emphasizing the crucial role of the trehalose pathway in the regulation of respiratory and fermentative metabolism., This work was supported, in part, by the ANR (Agence Nationale de la Recherche) Blanc [grant number 05-2-42128] and by the Region Midi Pyrénées (France) [grant number 09003813]. C.G. was supported by the Spanish Ministry of Science and Innovation (MICINN) [grant number BFU2010-19628-CO2-02]. A.U. thanks the HEC (Higher Education Commission) Pakistan for funding.

International audience; The recovery at a range of incubation temperatures and pH of spores of Bacillus weihenstephanensis KBAB4 exposed to a peracetic acid-based disinfectant (PABD) or to pulsed light was estimated. Spores of B. weihenstephanensis were produced at 30 °C and pH 7.00, at 30 °C and pH 5.50, or at 12 °C and pH 7.00. The spores were treated with a commercial peracetic acid-based disinfectant at 80 mg·mL−1 for 0 to 200 min at 18 °C or by pulsed light at fluences ranging between 0.4 and 2.3 J·cm−2 for pulsed light treatment. After each treatment, the spores were incubated on nutrient agar at 12 °C, 30 °C or 37 °C, or at pH 5.10, 6.00 or 7.40. Incubation temperature during recovery had a significant impact only near the recovery limits, beyond which surviving spores previously exposed to a PABD or to pulsed light were not able to form colonies. In contrast, a decrease in pH of the recovery nutrient agar had a progressive impact on the ability of spores to form colonies. The time to first log reduction after PABD treatment was 29.5 ± 0.7 min with recovery at pH 7.40, and was tremendously shortened 5.1 ± 0.2 min with recovery at pH 5.10. Concerning the fluence necessary for the first log reduction, it was 1.5 times higher when the spores were recovered at pH 6.00 compared to a recovery at pH 5.10. The impact of recovery temperature and pH can be described with a mathematical model using cardinal temperature and pH asparameters. These effects of temperature and pH on recovery of Bacillus weihenstephanensis spores exposed to a disinfectant combining peracetic acid and hydrogen peroxide, or pulsed light are similar, although these treatments are of different natures. Sporulation temperature or pH did not impact resistance to the peracetic acid-based disinfectant or pulsed light.

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